The dual PI3K/mTOR inhibitor PKI-587 enhances sensitivity to cetuximab in EGFR-resistant human head and neck cancer models.

BACKGROUND: Cetuximab is the only targeted agent approved for the treatment of head and neck squamous cell carcinomas (HNSCC), but low response rates and disease progression are frequently reported. As the phosphoinositide 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) pathways have an important role in the pathogenesis of HNSCC, we investigated their involvement in cetuximab resistance. METHODS: Different human squamous cancer cell lines sensitive or resistant to cetuximab were tested for the dual PI3K/mTOR inhibitor PF-05212384 (PKI-587), alone and in combination, both in vitro and in vivo. RESULTS: Treatment with PKI-587 enhances sensitivity to cetuximab in vitro, even in the condition of epidermal growth factor receptor (EGFR) resistance. The combination of the two drugs inhibits cells survival, impairs the activation of signalling pathways and induces apoptosis. Interestingly, although significant inhibition of proliferation is observed in all cell lines treated with PKI-587 in combination with cetuximab, activation of apoptosis is evident in sensitive but not in resistant cell lines, in which autophagy is pre-eminent. In nude mice xenografted with resistant Kyse30 cells, the combined treatment significantly reduces tumour growth and prolongs mice survival. CONCLUSIONS: Phosphoinositide 3-kinase/mammalian target of rapamycin inhibition has an important role in the rescue of cetuximab resistance. Different mechanisms of cell death are induced by combined treatment depending on basal anti-EGFR responsiveness.

Inhibition of Hedgehog signalling by NVP-LDE225 (Erismodegib) interferes with growth and invasion of human renal cell carcinoma cells.

BACKGROUND: Multiple lines of evidence support that the Hedgehog (Hh) signalling has a role in the maintenance and progression of different human cancers. Therefore, inhibition of the Hh pathway represents a valid anticancer therapeutic approach for renal cell carcinoma (RCC) patients. NVP-LDE225 is a Smoothened (Smo) antagonist that induces dose-related inhibition of Hh and Smo-dependent tumour growth. METHODS: We assayed the effects of NVP-LDE225 alone or in combination with everolimus or sunitinib on the growth and invasion of human RCC models both in vitro and in vivo. To this aim, we used a panel of human RCC models, comprising cells with acquired resistance to sunitinib - a multiple tyrosine kinase inhibitor approved as a first-line treatment for RCC. RESULTS: NVP-LDE225 cooperated with either everolimus or sunitinib to inhibit proliferation, migration, and invasion of RCC cells even in sunitinib-resistant (SuR) cells. Some major transducers involved in tumour cell motility, including paxillin, were also efficiently inhibited by the combination therapy, as demonstrated by western blot and confocal microscopy assays. Moreover, these combined treatments inhibited tumour growth and increased animal survival in nude mice xenografted with SuR RCC cells. Finally, lung micrometastasis formation was reduced when mice were treated with NVP-LDE225 plus everolimus or sunitinib, as evidenced by artificial metastatic assays. CONCLUSIONS: Hedgehog inhibition by NVP-LDE225 plus sunitinib or everolimus bolsters antitumour activity by interfering with tumour growth and metastatic spread, even in SuR cells. Thus, this new evidence puts forward a new promising therapeutic approach for RCC patients.

Toll-like receptor 9 agonist IMO cooperates with everolimus in renal cell carcinoma by interfering with tumour growth and angiogenesis.

BACKGROUND: Targeting the mammalian target of rapamycin by everolimus is a successful approach for renal cell carcinoma (RCC) therapy. The Toll-like receptor 9 agonist immune modulatory oligonucleotide (IMO) exhibits direct antitumour and antiangiogenic activity and cooperates with both epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) inhibitors. METHODS: We tested the combination of IMO and everolimus on models of human RCC with different Von-Hippel Lindau (VHL) gene status, both in vitro and in nude mice. We studied their direct antiangiogenic effects on human umbilical vein endothelial cells. RESULTS: Both IMO and everolimus inhibited in vitro growth and survival of RCC cell lines, and their combination produced a synergistic inhibitory effect. Moreover, everolimus plus IMO interfered with EGFR-dependent signaling and reduced VEGF secretion in both VHL wild-type and mutant cells. In RCC tumour xenografts, IMO plus everolimus caused a potent and long-lasting cooperative antitumour activity, with reduction of tumour growth, prolongation of mice survival and inhibition of signal transduction. Furthermore, IMO and everolimus impaired the main endothelial cell functions. CONCLUSION: A combined treatment with everolimus and IMO is effective in VHL wild-type and mutant models of RCC by interfering with tumour growth and angiogenesis, thus representing a potentially effective, rationale-based combination to be translated in the clinical setting.

The tissue-specific pathways regulating cell proliferation are inherited independently in somatic hybrid between thyroid and liver cells.

Thyroid stimulating hormone (TSH) and insulin-like growth factors type 1 (IGF-I) regulate the proliferation and differentiation of cultured thyroid cells but not of cultured liver cells. We have examined the influence of TSH and IGF-I on the metabolic functions and proliferation of somatic hybrids obtained by fusing rat thyroid cells (FRTL5) with rat liver cells (BRL). While IGF-I is able to stimulate the proliferation of the hybrid cells (TxL) TSH fails to induce their growth. However, the hybrid TxL cells have surface TSH receptors with normal ligand characteristics. The addition of TSH to TxL cells led to typical enhancement of cAMP production and depolymerization of actin filaments. Yet, TSH failed to stimulate iodine uptake in the hybrid cells. Interestingly, iodine inhibited TxL proliferation induced by IGF-I but not by serum. It is concluded that the hybrid TxL cells inherited from the parental thyroid cells several important differentiated traits including mitogenic pathways induced and used by IGF-I, functional TSH receptors, and sensitivity to the inhibitory action of iodine.

Oral administration of a novel taxane, an antisense oligonucleotide targeting protein kinase A, and the epidermal growth factor receptor inhibitor Iressa causes cooperative antitumor and antiangiogenic activity.

PURPOSE: Protein kinase A type I (PKAI) and the epidermal growth factor receptor (EGFR) play a role in neoplastic transformation and interact with each other in transducing mitogenic signals. We developed different PKAI and EGFR inhibitors, demonstrating their cooperation with cytotoxic drugs and the therapeutic potential of the combined blockade of PKAI and EGFR. In this study, we investigated the effect of orally active PKAI and EGFR inhibitors in combination with a novel taxane. EXPERIMENTAL DESIGN: We combined a hybrid PKAI antisense oligonucleotide sequence (AS-PKAI), the EGFR inhibitor ZD1839 (Iressa), and the taxane IDN5109, studying their effect on human cancer growth, apoptosis, and angiogenesis and measuring vascular endothelial growth factor (VEGF) expression and vessel formation in vitro and after oral administration in nude mice. RESULTS: We demonstrated cooperative growth inhibitory and proapoptotic effects and inhibition of VEGF expression with any combination of two drugs and a marked synergistic effect when all three agents were combined. Oral administration of AS-PKAI, ZD1839, and IDN5109 in combination to nude mice caused a remarkable antitumor effect with no histological evidence of tumors in 50% of mice 5 weeks after treatment withdrawal, accompanied by complete suppression of vessel formation and VEGF expression. CONCLUSION: This is the first demonstration of the cooperative antitumor and antiangiogenic activity of three novel agents that block multiple signaling pathways after oral administration. Because all agents are under clinical evaluation in cancer patients, our results provide a rationale to translate this feasible therapeutic strategy in a clinical setting.

Adenosine 3',5'-monophosphate mediates both the mitogenic effect of thyrotropin and its ability to amplify the response to insulin-like growth factor I in FRTL5 cells.

In previous studies we have demonstrated that bovine TSH (bTSH) and insulin-like growth factor I (IGF-I) independently stimulate both the incorporation of [3H]thymidine into DNA and replication in quiescent FRTL5 cells. In the case of TSH, evidence was presented that these responses are cAMP mediated. In addition, responses of thymidine incorporation are greatly amplified when particular concentrations of the two agents are added together, but this effect diminishes as the concentration of either bTSH or IGF-I is increased. The present experiments were undertaken to obtain further information concerning the mechanism of the independent mitogenic effects of bTSH and IGF-I and to explore the nature of the biphasic synergistic interaction with respect to thymidine incorporation that occurs when bTSH and IGF-I are added together. Verification that the increases in [3H] thymidine incorporation induced by bTSH and IGF-I, alone and together, are truly reflective of increases in DNA synthesis was obtained in experiments in which labeled nuclei were counted in cultures of cells grown in the presence of one or both mitogenic agents to which [3H]thymidine had been added. In these studies the number of cells with labeled nuclei was increased markedly by each of the two agents, and the response when the two mitogens were added together was far greater than the sum of their individual effects. Over a range of concentrations which included those that elicit a mitogenic response in FRTL5 cells, IGF-I, unlike bTSH, failed to increase cAMP generation when added alone. Moreover, IGF-I did not significantly enhance the cAMP response to varying concentrations of bTSH. A concentration-dependent increase in the incorporation of [3H]thymidine into DNA was induced by culturing cells in the presence of the cAMP analog (Bu)2cAMP (Bt2cAMP), the phosphodiesterase inhibitor isobutylmethylxanthine, and the stimulator of adenylate cyclase forskolin. When increasing concentrations of these agents were added together with IGF-I, a biphasic pattern of response of DNA synthesis, mimicking that produced by the combination of IGF-I and increasing concentrations of bTSH, was observed. Further evidence that cAMP mediates the mitogenic response to bTSH was the observation that adenosine inhibited the stimulation of both cAMP generation and DNA synthesis that bTSH produced. Although preincubation of quiescent FRTL5 cells for 24 h in the presence of bTSH resulted in only a small increase in DNA synthesis, measured during the last 3 h of a subsequent 24-h incubation carried out in the absence of bTSH, it greatly amplified the response to IGF-I added alone during the second incubation.(ABSTRACT TRUNCATED AT 400 WORDS)

Independent and interactive effects of tetradecanoyl phorbol acetate on growth and differentiated functions of FRTL5 cells.

In studies of regulation of the growth and differentiated function of the thyroid follicular cell, we have employed the FRTL5 cell line to evaluate both the effects of agents that activate protein kinase-C (PKC) and their interaction with other agents that influence the growth and/or function of the FRTL5 cell. The PKC activator tetradecanoyl-phorbol acetate (TPA) alone induced a time- and concentration-dependent stimulation of the incorporation of [3H]thymidine into the DNA of quiescent FRTL5 cells, an effect anteceded by an increase in the levels of the mRNAs of the proto-oncogene c-myc and associated with a stimulation of cell replication. TPA also produced a dose-dependent inhibition of the low levels of radioiodine uptake in quiescent FRTL5 cells. These effects of TPA were unaccompanied by any change in the cellular cAMP concentration. TPA also modified a variety of responses to TSH, attenuating the TSH-induced stimulation of [3H]thymidine incorporation into DNA, cell replication, cAMP generation, and iodine uptake. Inhibition of TSH-stimulated growth and iodine uptake by TPA could not be ascribed solely to a decrease in cAMP generation, since TPA also inhibited the increase in [3H]thymidine incorporation and iodide uptake induced by the cAMP analog (Bu)2cAMP. In contrast, the independent stimulatory effects of TPA and insulin-like growth factor I (IGF-I) on [3H]thymidine incorporation and cell replication were at least additive when the two stimulators were added together. We have previously reported that both TSH and (Bu)2cAMP amplify the enhancement of DNA synthesis and cell replication in FRTL5 cells induced by IGF-I, and that the response of DNA synthesis to IGF-I is also enhanced if cells are preincubated with either TSH or (Bu)2cAMP. Both the former amplification of mitogenesis and the latter priming effect were decreased by exposing cells to TPA concomitant with their exposure to TSH or (Bu)2cAMP. The effects of TPA were mimicked by other activators of PKC, but not by a phorbol ester that fails to activate this enzyme. In general, we conclude that in the FRTL5 cell, regulation of cell growth is extremely complex; there are at least three mitogenic pathways that are separate from but interact with one another. The first is the well known cAMP-dependent pathway, which is activated by TSH. The second is activated by IGF-I and is cAMP independent. These two pathways interact to produce a marked amplification of their individual mitogenic effects. The third pathway is that stimulated by TPA and involves activation of PKC.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine has divergent effects on deoxyribonucleic acid synthesis in FRTL5 cells: inhibition of thyrotropin-stimulated and potentiation of insulin-like growth factor-I-stimulated thymidine incorporation.

Adenosine inhibits TSH-stimulated [3H]thymidine incorporation into DNA in FRTL5 thyroid follicular cells by both inhibiting cAMP generation and acting at a locus beyond adenylate cyclase. On the other hand, adenosine markedly potentiates DNA synthesis in FRTL5 stimulated by insulin-like growth factor-I (IGF-I). The mechanisms of this latter effect are unknown, but require the coincubation of adenosine and IGF-I and not mediated by an increase in intracellular cAMP concentration. Adenosine increases the maximal response of FRTL5 to [3H]thymidine incorporation stimulated by IGF-I and increases the sensitivity of FRTL5 to IGF-I. These effects of adenosine are reflected by an increase in nuclear labeling as well as by an increase in [3H]thymidine incorporation into DNA. Adenosine also plays a role as an autocrine growth factor in FRTL5, since adenosine deaminase increases the response of these cells to TSH. The effects of adenosine on both TSH- and IGF-I-stimulated DNA synthesis are shared by guanosine and inosine, although with different potencies for the various guanine nucleosides. Inosine potentiates IGF-I-stimulated DNA synthesis, but inhibits TSH-stimulated DNA synthesis only weakly. Adenosine interacts with multiple receptors and with multiple postreceptor pathways in FRTL5 to produce divergent effects on the control of cell replication by two growth factors (TSH and IGF-I) that act through different postreceptor pathways.

Iodine inhibits the proliferation of rat thyroid cells in culture.

We have explored the mechanisms whereby iodine inhibits thyroid growth using as models both the FRTL5 line of rat thyroid follicular cells that require TSH for growth and the M12 line of mutant cells that grow in the absence of TSH. Between 0.01-1.0 mM, NaI produced a dose-dependent inhibition of TSH stimulation of [3H]thymidine incorporation and replication in FRTL5 cells as well as spontaneous growth in M12 cells. Iodide also inhibited the cAMP-dependent growth of FRTL5 cells induced by forskolin and (Bu)2cAMP, as well as the cAMP-independent mitogenesis induced by insulin-like growth factor-I. The effect of iodide to inhibit both TSH- and insulin-like growth factor-I-stimulated growth in FRTL5 cells was abolished by concomitant culture with methimazole, and no iodide inhibition of growth was observed in L6 myoblasts and BRL 30E hepatocytes. Exposure of cells to iodide under conditions that resulted in inhibition of TSH-stimulated growth did not significantly alter the ability of TSH to increase the intracellular cAMP concentration, nor did iodide alter two responses to TSH in FRTL5 cells that depend upon an increase in cAMP concentration: down-regulation of TSH receptor and cytoskeletal reorganization. We conclude that iodide exerts its inhibitory action on the growth of thyroid cells at multiple loci related to both the cAMP-dependent and cAMP-independent pathways of mitogenic regulation.

The disulfide bond pattern between fragments obtained by the limited proteolysis of bovine thyroglobulin.

The comparative analysis of the products of the limited proteolysis of bovine thyroglobulin with trypsin by SDS-polyacrylamide gel electrophoresis in non-reducing and reducing conditions revealed the presence of disulfide linkages between some of the fragments. In order to define the disulfide bond pattern between the proteolytic fragments of thyroglobulin, these were isolated by SDS-polyacrylamide gel electrophoresis in non-reducing conditions and electrophoretic transfer onto polyvinylidene difluoride membranes. Individual bands were desorbed from the membranes and re-analyzed by SDS-polyacrylamide gel electrophoresis in reducing conditions. The resulting peptides were identified by comparison with the peptides directly obtained by SDS-electrophoresis in reducing conditions, and characterized by amino-terminal peptide sequencing either in this study or in a previous investigation (Gentile F., Salvatore G., Eur. J. Biochem. 218 (1993) 603-621). The analysis revealed that several fragments, produced by cleavages within the context of various cysteine-rich repeats of type 1 and within cysteine-rich repeat 3b.1, did not separate in the absence of reduction. On the other hand, the products of the cleavages at the carboxy-terminal extremity of the linker between type 2 and type 3 cysteine-rich repeats, and in the middle of the acetylcholinesterase-similar domain of thyroglobulin separated freely, with no need for reduction. On the base of these data, a model is presented in which distinct subsets of cysteine-rich repeats and the carboxy-terminal, acetylcholinesterase-similar domain of thyroglobulin form sequentially aligned subdomains with internal disulfide linkages.

In the thyroid cells proliferation, differentiated and metabolic functions are under the control of different steps of the cyclic AMP cascade.

In the course of studies to elucidate the complex network of interactions controlling FRTL5 cell proliferation, thyroid stimulating hormone (TSH)-independent mutants (M cells), have been obtained from FRTL5 cells by chemical mutagenesis. In the present studies, the role of TSH on the proliferation and on differentiated and metabolic functions in these mutant cells have been investigated and compared to their response to insulin-like growth factor I (IGF-I). The addition of IGF-I to M cells leads to normal stimulation of DNA synthesis. However, inspite of the fact that mutant cells display normal TSH receptors, TSH is unable to stimulate the proliferation of the M cells. Nevertheless, TSH is able to increase intracellular levels of cAMP leading to regulation of TSH function in the M cells. On the other hand, TSH does not influence iodide transport and actin filaments depolimerization in these cells. However, aminoacid transport, stimulated in wild-type FRTL5 cells by both TSH and IGFs, is under the control of IGFs but not of TSH in the mutant cells. Neither TSH or IGF-I modified the expression of c-fos proto-oncogene in the M cells, probably because of high constitutive expression. These data suggest that a crucial signalling step(s) required for TSH induced mitogenesis is impaired in the M cells, and that this signalling step is not required for IGF-I induced mitogenesis.

Transfected insulin-like growth factor II modulates the mitogenic response of rat thyrocytes in culture.

Rat thyroid cells (FRTL5), transfected with the sequence coding for rat insulin-like growth factor II (IGF-II) presented mRNA specific for the transfected IGF-II in most of the clones obtained (Tr clones). Tr7 and Tr12 cells maintained their ability to respond to the mitogenic effect of thyrotropin (TSH), while either exogenous IGF-I or IGF-II or insulin failed to stimulate their proliferation. In the absence of exogenous mitogens the Tr7 and Tr12 clones vigorously incorporated [3H]thymidine into DNA. This activity was significantly inhibited by sm1.2, a monoclonal antibody against rat IGF-II. Tr7 and Tr12 clones possess type I IGF receptors, known to mediate the mitogenic effect of IGF-II, with affinity similar to those present on the membrane of the parental cells but with reduced capacity. Finally, media conditioned by Tr7 and Tr12 increase basal thymidine incorporation in quiescent FRTL5 cells and amplify that induced by TSH. Endogenous IGFs may play an important role in the regulation of thyroid cell proliferation by modulating the mitogenic effect of TSH and by supporting TSH-independent growth.

Polyacrylamide gel electrophoresis in discontinuous transverse urea-gradient gels.

Polyacrylamide gel electrophoresis in transverse urea-gradient gels is widely used for the study of changes in conformation and quaternary structure of proteins induced by increasing concentrations of urea. However, possible uncertainties regarding the effective concentration of urea at any given point across the gel must be considered when this technique is used for quantitative purposes. We describe here a simple, practical procedure for preparing urea-gradient gels discontinuously, by stacking various layers of acrylamide solution with increasing urea concentrations. We show that the urea concentration in different lanes of discontinuous gradient gels prepared in this way can be predetermined accurately enough for quantitative purposes and provide as an example the study of the dissociation of thyroglobulin in urea. Various levels of resolution can be attained by this technique. The use of discontinuous gradient gels may extend the usefulness of urea gel electrophoresis in relation with the quantitative aspects of the study of protein conformation.

Expression of the soluble lectin L-14 gene is induced by TSH in thyroid cells and suppressed by retinoic acid in transformed neural cells.

Expression of the soluble lectin L-14 is low in normal and very high in transformed thyroid cells. We show that L-14 gene expression is transiently induced upon thyrotropin stimulation of normal quiescent FRTL-5 rat thyroid cells. Permanent activation of L-14 gene expression is obtained in the same cells infected with a wild-type and a temperature sensitive mutant of Kirsten murine sarcoma virus, both at the permissive and non permissive temperature for transformation. We also find that L-14 mRNA is undetectable in rat brain but is abundant in rat oligodendrocytes precursors transformed by polyoma middle T oncogene. Retinoic acid treatment of these transformed cells leads to acquisition of a differentiated phenotype accompanied by a 30-fold decrease of L-14 mRNA levels. Removal of retinoic acid restores both the transformed undifferentiated phenotype and high L-14 expression. Taken together these results indicate that growth stimulation and induction of cell differentiation are accompanied by strong modulation of L-14 gene expression.

Sustained versus transient cyclic AMP intracellular levels: effect on thyrotropin-dependent growth of thyroid cells.

Cyclic AMP (cAMP) is the second messenger that stimulates growth and differentiation of thyroid cells, which are dependent upon thyrotropin for the initiation of the cell cycle. Treatment of thyroid cells with phosphodiesterase inhibitors, such as 1-methyl-3-isobutylxanthine (IBMX), RO-20-1724, or aminophylline, induces persistent levels of cAMP and blocks cell proliferation. IBMX-treated cells are arrested at the G1-S border, but removal of the drug allows cell growth to resume. The inhibiting effect of IBMX is dose dependent, and the phase of the cell cycle is irrelevant. These data indicate that prolonged and steady accumulation of cAMP blocks the cell cycle in thyroid cells.